The present invention relates to a process for purification of cyclopeptide compounds containing at least one protonatable amino group, in particular, the process relates to the purification of an Echinocandin-type compound by adsorption onto a hydrophobic, reversed phase chromatographic media and eluting with a continuous nearly linear gradient of increasing acetic acid. A purification process is also provided for selectively removing a tripeptide-aldehyde by-product of the Echinocandin fermentation process to yield a higher purity Echinocandin compound.
Echinocandin cyclopeptides are natural products that have been shown to have antifungal activities. Included in the Echinocandin cyclopeptide family are natural products such as Echinocandin B (ECB), Echinocandin C, Aculeacin Axcex3y, Mulundocandin, Sporiofungin A, Pneumocandin A0, WF11899A, and Pneumocandin B0. The natural products are typically produced by culturing various microorganisms. For example, Echinocandin B is produced from the fermentation of the fungus, Aspergillus nidulans. 
In the search for more active materials, the natural products have been modified in a variety of ways. One of the most common modifications has been the replacement of the N-acyl side chain on the natural product to produce a semi-synthetic derivative. For example, U.S. Pat. Nos. 4,293,489; 4,320,052; 5,166,135; and 5,541,160; and EP 359529; 448353; 447186; 462531; and 561639 describe a variety of N-acyl derivatized Echinocandin compounds that provide varying degrees of antifungal activity.
The N-acyl derivatives are produced by deacylating the natural product followed by reacylation with a different acyl group. The deacylation is typically achieved by means of an enzyme (e.g., deacylase enzyme). The deacylase enzyme may be obtained from the microorganism Actinoplanes utahensis or Pseudomonas species. See i.e., U.S. Pat. Nos. 4,293,482; and 4,304,716; and EP 460,882. The deacylated compound is typically referred to as the nucleus of the corresponding natural product (i.e., the deacylated product of Echinocandin B is referred to as the Echinocandin B nucleus (ECBN)). Unfortunately, both the fermentation and deacylation processes produce several by-products that are difficult to remove and decrease the purity of the desired deacylated cyclic peptide nucleus.
U.S. Pat. No. 4,874,843 describes a chromatographic process using non-functional resins in a reversed mode to purify Echinocandin-type products. Even though the process improved the purity of products derived from a fermentation process, further improvements are still needed to remove contaminants that are difficult to separate from both the intermediate deacylated nucleus and the final acylated pharmaceutically active compounds. Since the potency of the final pharmaceutical product is dependent upon the purity of the intermediates used to make the final product, improvements in purity at any stage of the manufacturing process are highly desirable. Ideally, the contaminants are removed at the earliest stage possible in the manufacturing process.
General discussions of non-functional resins and their applications in liquid chromatographic separations may be found in J. Chromatography, 201, 287-292 (1980) and Grieser, M.D. et al, Analytical Chemistry, 45, 1348-1353 (1973). The use of either step or continuous gradients are discussed; however, the eluents contain significant amounts of organic solvents. In a manufacturing process, the use of organic solvents raises several concerns such as environmental regulations (e.g., air quality emission standards), special handling requirements (e.g., flammability standards) and disposal limitations (e.g., toxic waste regulations). Therefore, there is a need for an eluent system that minimizes the use of organic solvents yet effectively separates mixtures into their pure components.
The present invention provides a method for separating and purifying a wide variety of fermentation cyclopeptide products containing at least one protonatable amino group (including the deacylated Echinocandin-type compounds) from their fermentation or mixed broths and partially purified process streams by adsorbing the mixture onto a hydrophobic, reversed phase, chromatographic media and eluting with a continuous nearly linear acetic acid gradient ranging from 0.1% acetic acid to 10.0% acetic acid by volume in water, preferably from 0.5% (pH=5.5) to 4.0% (pH=2.5) acetic acid.
In another embodiment of the present invention, a process for purifying Echinocandin-type compounds (including simple derivatives thereof) is provided where an aldehyde by-product (in particular, a tripeptide-aldehyde by-product) in the fermentation mixture or partially purified mixture is reacted with a derivatizing agent. Preferably, the fermentation broth or mixed broth is reacted with the derivatizing agent prior to purification of the corresponding Echinocandin nucleus using the method described above.
As used herein, the term xe2x80x9cderivatizing agentxe2x80x9d refers to a reagent capable of reacting with the aldehyde functionality of the tripeptide by-product to produce an intermediate that is sufficiently different in hydrophobicity to allow separation of the tripeptide intermediate from the desired Echinocandin-type compound.
The term xe2x80x9cprotonatable amino groupxe2x80x9d refers to an amino group that undergoes protonation when subjected to the eluting conditions of the present invention (i.e., 0.1% acetic acid to 10% acetic acid by volume in water).
The term xe2x80x9cEchinocandin-type compoundsxe2x80x9d refers to compounds having the following general structure including any simple derivatives thereof: 
wherein R is a hydrogen or xe2x80x94C(O)Rxe2x80x2 where Rxe2x80x2 is an alkyl group, an alkenyl group, an alkynyl group, an aryl group, or heteroaryl group having attached thereon at least one protonatable amino group; R1 is xe2x80x94H or xe2x80x94OH; R2 is xe2x80x94H or xe2x80x94CH3; R3 is xe2x80x94H, xe2x80x94CH3, xe2x80x94CH2CONH2 or xe2x80x94CH2CH2NH2; R4 is xe2x80x94H or xe2x80x94OH; R5 is xe2x80x94OH, xe2x80x94OPO3H2, or xe2x80x94OSO3H; and R6 is xe2x80x94H or xe2x80x94OSO3H. xe2x80x9cEchinocandin nucleusxe2x80x9d refers to the deacylated Echinocandin compound where R is a hydrogen. xe2x80x9cECBNxe2x80x9d refers to the Echinocandin B nucleus where R1, R4 and R5 are hydroxyl groups, R2, R3, and R7 are methyl groups; and R1 and R6 are hydrogens.
The term xe2x80x9calkylxe2x80x9d refers to a hydrocarbon radical of the general formula CnH2n+1 containing from 1 to 30 carbon atoms unless otherwise indicated. The alkane radical may be straight (e.g. methyl, ethyl, propyl, butyl, etc.), branched (e.g., isopropyl, isobutyl, tertiary butyl, neopentyl, etc.), cyclic (e.g., cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl, etc.), or multi-cyclic (e.g., bicyclo[2.2.1]heptane, spiro[2.2]pentane, etc.). The alkane radical may be substituted or unsubstituted. Similarly, the alkyl portion of an alkoxy group or alkanoate has the same definition as above.
The term xe2x80x9calkenylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon-carbon double bond. The alkene radical may be straight, branched, cyclic, or multi-cyclic. The alkene radical may be substituted or unsubstituted.
The term xe2x80x9calkynylxe2x80x9d refers to an acyclic hydrocarbon containing at least one carbon-carbon triple bond. The alkene radical may be straight, or branched. The alkyne radical may be substituted or unsubstituted.
The term xe2x80x9carylxe2x80x9d refers to aromatic moieties having single (e.g., phenyl) or fused ring systems (e.g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups may be substituted or unsubstituted.
The term xe2x80x9cheteroarylxe2x80x9d refers to aromatic moieties containing at least one heteratom within the aromatic ring system (e.g., pyrrole, pyridine, indole, thiophene, furan, benzofuran, imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic moiety may consist of a single or fused ring system. The heteroaryl groups may be substituted or unsubstituted.
Within the field of organic chemistry and particularly within the field of organic biochemistry, it is widely understood that significant substitution of compounds is tolerated or even useful. In the present invention, for example, the term alkyl group allows for substitutents which is a classic alkyl, such as methyl, ethyl, propyl, hexyl, isooctyl, dodecyl, stearyl, etc. The term xe2x80x9cgroupxe2x80x9d specifically envisions and allows for substitutions on alkyls which are common in the art, such as hydroxy, halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as well as including the unsubstituted alkyl moiety. However, it is generally understood by those skilled in the art that the substituents should be selected so as to not adversely affect the pharmacological characteristics of the compound or adversely interfere with the use of the medicament. Suitable substituents for any of the groups defined above include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and di-alkyl amino, quaternary ammonium salts, aminoalkoxy, hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl, carboxy, glycolyl, glycyl, hydrazino, guanyl, and combinations thereof.
Fermentation and mixed broths contain a number of by-products that are very difficult to separate from the desired cyclopeptide product. xe2x80x9cMixed brothxe2x80x9d refers to a conversion mixture where the fermentation broth is treated directly with a deacylating enzyme without purification to produce the deacylated product (e.g. ECBN). Reversed-phase, liquid chromatography has been used in the past with reasonable success; however, the need for higher purity compounds demands more improved methods of purification. Applicants have discovered that the separation of fermentation by-products from the desired fermentation product containing a protonatable amino group can be improved by using a reversed-phase chromatographic media in combination with a continuous, nearly linear acetic acid elution scheme.
Suitable hydrophobic chromatographic media include reversed phase silicas, and organic polymers such as copolymers of styrene and divinylbenzene, methacrylate polymer. A variety of reversed-phase silicas are commercially available from vendors such as BTR, E. Merck, Eka Nobel, Millipore, Phenomenex, Whatman, or YMC. The silicas are derivatized with straight chain alkyl hydrocarbons ranging in length from C1 to C18 (C1, C4, C8 and C18 being the most common) or other hydrophobic ligands such (e.g. phenyl or cyano). A variety of styrene/divinylbenzene resins designed for reversed-phase, liquid chromatography are also available commercially such as Diaion(trademark) HP and SP resins (available from Mitsubishi Chemical Industries Limited, Tokyo, Japan), and Amberlite XAD-2,4, and 16 resins (available from Rohm and Haas Chemical Co., Philadelphia, Pa.), and the CG-161, 300, and 1000 Amberchrom resins from Toso Haas (Montgomeryville, Pa.). Non-functional resins are generally characterized by their pore volume (0.5-4.5 ml/g), specific surface area (200-800 m2/g), pore diameter (40-1300 xc3x85), pore size distribution and/or bead size distribution. Preferred non-functional resins include Diaion HP-20 having a surface area of 500 m2/g, a pore size of 200-300 xc3x85 and particle size of 200-800 xcexcm; SP-825 having a surface area of 1,000 m2/g, pore size of 50-60 xc3x85 and particle size of 250-600 xcexcm; SP-207 (brominated version of HP-20) having a surface area of 630 m2/g, pore size of 100-200 xc3x85 and particle size of 200-800 xcexcm; and CG-161CD having a surface area of 900 m2/g, pore size of 110-175 xc3x85, and particle size of 80-160 xcexcm. More preferred are the HP-20 and SP-825 resins.
Initially, a crude or partially purified solution is provided that contains the desired cyclic peptide compound having at least one protonatable amino group. Generally, the amino group can be protonated during the course of the acetic acid gradient that spans the pH range from 5.5 to 2.5. Preferably the amino group is a primary amine; however, the amino group may be a secondary amine or tertiary amine so long as the additional substituents on the nitrogen atom are not sufficiently hydrophobic such that they overcome the polarity of the positively charged amine. The solution may originate from a fermentation process or a synthetic process. For example, the cyclic peptide compounds may be prepared by the synthetic methods described in U.S. Pat. No. 5,696,084; J. Am. Chem. Soc., 108, 6041 (1986); Evans, D. A., et al., J. Am. Chem. Soc., 109, 5151 (1987); J. Med. Chem., 35, 2843 (1992); and Kurokawa, N., et al., Tetrahedron, 49, 6195 (1993). The crude solution is usually a mixed broth. Alternatively, the process may be used to further purify (or polish) partially purified material.
Depending upon the particular fermentation process used, it may be desirable to prefilter the solution to remove particulates that may interfere with the chromatographic process. Filtration may be accomplished by any number of means known to those skilled in the art including gravity filtration, vacuum filtration through a ceramic filter which may or may not include a Celite(trademark) filter aid, etc. Solids in the fermentation broth may also be removed by centrifugation followed by decanting the liquid from the solids.
The fermentation solution may be concentrated if desired using a variety of means which are also well known to those skilled in the art such as evaporative concentration, lyophilization, etc. The concentrate may be filtered a second time to remove any precipitate that may have formed during the concentration process.
The crude or partially purified solution is loaded onto a chromatography column packed with one of the hydrophobic chromatographic media described above. The desired cyclic peptide product is then eluted from the chromatographic media using a continuous nearly linear gradient ranging from about 0.1% acetic acid to about 10% acetic acid, preferably from about 0.5% acetic acid (pH=5.5) to about 4% acetic acid (pH=2.5). The upper end of the range of acetic acid concentration selected is based upon the stability of the chromatographic media used and the stability of the compound being purified at that pH. The lower end of the range is selected based upon the pH where the amino group is protonated and concentration of acetic acid required to elute the product from the hydrophobic surface. Those skilled in the art will appreciate that the gradient does not have to be perfectly linear. Within the meaning of xe2x80x9cnearly linearxe2x80x9d includes a flat convex or concave gradient.
At the end of the gradient elution process step, an additional volume of the higher concentrated acetic solution is typically used to complete the elution. At the end of the process, the column may be regenerated so that the column may be re-used for additional purification cycles. The regeneration step typically involves washing the column with mixtures of an organic solvent and water at both a neutral and alkaline pH to remove any residual materials left on the column matrix. Suitable solvents include acetonitrile, methanol, isopropanol, and acetone. The linear acetic acid elution scheme not only provides good selectivity (see i.e., Example 1 below), but also limits the use of organic solvents to the regeneration step of the column operation. Thus, both the absolute quantity of organic solvent used and the volume of column effluent that must be treated prior to disposal is minimized.
The fermentation product may be recovered from the eluate using a variety of methods. Suitable recovery methods include crystallization, evaporative concentration, and lyophilization.
The fermentation broth for Echinocandin B contains varying levels of a tripeptide-aldehyde (Asn-Gln-Leu-H) by-product having the following chemical structure (Ia). The tripeptide-aldehyde by-product under goes deacylation as well as Echinocandin B during the enzymatic deacylation process to form the corresponding deacylated tripeptide-aldehyde (Ib). 
where R is C(O)CH2CH(OH)C9H19 (Iaxe2x80x94fermentation by-product) or a hydrogen (Ibxe2x80x94deacylation by-product from a mixed broth).
Surprisingly, the retention time of the deacylated tripeptide-aldehyde is very similar to ECBN in reversed phase, liquid chromatography, even under optimum elution conditions, thus making it very difficult to separate the deacylated tripeptide-aldehyde (Ib) from the desired ECBN. If the tripeptide is not removed, then the free amino group of the tripeptide competes with the free amino group of the ECBN compound during the reacylation process. As a result, an excess of acylating compound must be added to insure complete acylation of the ECBN compound. The tripeptide contaminate not only consumes needed starting materials but also produces an acylated by-product that is difficult to remove in subsequent purification of the acylated ECB compound. Preferably, the tripeptide by-product is removed prior to the reacylation of the ECBN compound.
The tripeptide-aldehyde by-product may be removed from either the fermentation mixture or the mixed broth (i.e., deacylation mixture) by reacting the aldehyde with a derivatizing agent prior to chromatographic purification. The derivatizing agent may be added to the aldehyde functionality to change the chromatographic retention time of the tripeptide-aldehyde in relation to the desired ECB compound. Suitable derivatizing agents include sodium bisulfite, hydroxyl amine and semicarbazide hydrochloride. Some advantages of using derivatizing agents as opposed to other means of modifying the chromatographic retention times are the selectivity of the derivatizing agents for the aldehyde functionality and the mild conditions under which the reaction occurs. If the reaction between the derivatizing agent and the tripeptide-aldehyde is reversible, then the aldehyde can be easily recovered by removing the derivatizing agent. The recovered tripeptide can then be used for other purposes.